中性粒细胞在肺血栓栓塞症犬肺缺血-再灌注损伤中的作用

The role of polymorphonuclear cells in lung ischemia-reperfusion injury in a canine model of pulmonary thromboembolism

目的观察中性粒细胞(PMN)在肺血栓栓塞症(PTE)犬肺缺血-再灌注(I/R)损伤中的作用。方法 15只犬按随机数字表法随机分为3组:假手术组、缺血组、再灌注组。对 PTE 犬行取栓术,分别观察肺组织普通病理、组织匀浆髓过氧化物酶(MPO)含量及超微结构;Annexin V-FITC 凋亡检测试剂盒标记后用流式细胞仪检测再灌注前及再灌注后2、4、6 h 外周血 PMN 的凋亡率。结果再灌注6 h 后,再灌注组肺泡腔 PMN 计数明显高于假手术组及缺血组[(31±11)、(1±1)及(8±4)/10个高倍镜视野],肺组织匀浆 MPO 含量分别为(11.7±1.6)、(8.3±2.1)及(9.1±0.5)μg/L;缺血组肺泡腔内少许炎细胞渗出,再灌注组肺泡腔内见大量以 PMN 为主的炎细胞渗出;电镜下,再灌注后PMN 紧紧黏附于肺泡间隔,间隔内含水肿液及空泡样变性等;再灌注6 h 后外周血 PMN 凋亡率为(3.0±2.5)%,明显低于再灌注前的(7.4±4.5)%;术后4 h,再灌注组外周血 PMN 凋亡率为(4.8±2.6)%,明显低于缺血组的(9.3±2.0)%;6 h 组结果类似[(3.0±2.5)%,(8.0±1.6)%]。结论在该 PTE 犬 I/R 模型中,再灌注后 PMN 活性增强、凋亡减少;PMN 的参与是该模型肺 I/R 损伤的重要细胞学机制之一。

Objective To explore the effects of polymoronuclear cells （PMN） on lung ischemia- reperfusion（I/R） injury in a canine model of pulmonary thromboembolism. Methods Fifteen dogs were divided into three groups：a sham group （ n = 5 ）, an ischemia group （ n = 5 ） and a reperfusion group （ n = 5）. PMN in the whole blood were isolated with density gradient centrifugation. Apoptosis rates of the PMN was measured through flow cytometer after the cells were labeled by Annexin V-FITC before embolectomy and at 2, 4, 6 h after the operation. The myeloperoxidase （MPO） concentrations in lung homogenates were measured by ELISA. Alveolar PMN in the reperfusion lobar were observed by optical microscopy. The lung ultrastructure was studied by transmission electron microscopy. Results Alveolar PMN infiltration and the concentrations of MPO in the reperfusion group were significantly higher than those in the ischemia group （PMN （31 ±11） vs （8 ±4）/ ten high power fields, MPO （11.7 ± 1.6） vs （9. 1 ±0.5） μg/L, P 〈 0.05）. In the reperfusion group, abundant inflammatory cell infiltration was observed, predominantly with PMN in the alveoli. Apoptosis rates of the blood PMN at 6 h after reperfusion were much lower than before reperfusion （3. 0 ± 2. 5 ） % vs （7.4 ± 4. 5） %, P 〈 0. 05 ）. At 4 and 6 h after operation, the PMN apoptosis rates in the reperfusion group were significantly lower than the ischemia group （4 h ： （4. 8 ± 2. 6 ） % vs （9. 3 ±2.0）% ,6 h： （3. 0 ±2. 5）% vs （8.0 ± 1.6）%, P 〈0. 05）. PMN attaching firmly to the alveolar septum were observed by electron microscope. Conclusion PMN with enhanced activities and decreased apoptosis rate, are involved in the cellular mechanisms of the lung I/R injury in this model of pulmonary thromboembolism.